Fermentation of amylaceous materials



Patented Nov. 2, 1937 FERMENTATION OF MATER AMYLACEOUS IALS Hugh R. Stiles, Terre Haute, Ind., assignor to Commercial Solvents Corporation, Terre Hautc, Ind., a corporation of Maryland No Drawing. Application March 2, 1934,

Serial No. 713,692

14 Claims.

My invention relates to the production of butyl alcohol and other valuable products by the fermentation of starchy carbohydrate mashes. More specifically, my invention relates to the production of normal butyl alcohol, acetone, and ethyl alcohol by the fermentation of starchy mashes, and especially cereal mashes, by means of bacteria designated herein as Clostridium saccharo-acetobutylicum.

It has previously been known that starch mashes could be fermented by means of organ-'- isms of the Clostridium butyricum group with the production of various products such as acetic and butyric acids, butyl alcohol, etc. (see for example, Bergeys Manualof Determinative Bacteriology, Williams & Wilkins Co., Baltimore, 1925, pages 326-7) However, the yields of butyl alcohol have in practically all cases been so low as to preclude commercial utilization of such fermentations. This is particularly true in the case of cereal mashes such as corn mash. Up to the present time, therefore, the commercial production of butyl alcohol by fermentation has'been accomplished only by means of organisms of the type Clostridium acetobutylicum (Weizmann). I have now discovered that the group of bacteria designated herein as Clostridium saccharo-' acetobutylicum will produce satisfactory yields of butyl alcohol from starchy mashes such as corn mash, if there are maintained certain fermentation conditions, especially the provision of a suitable source of nitrogen, and a control of the hydrogen ion concentration by means of sub- So much stances such as calcium carbonate. confusion exists in the nomenclature and reported cultural characteristics of the prior art organisms of the Clostrz'dium butyricum type that it is impossible to state definitely .if any of them are included in the group now designated as 40 Clostridium saccharo-acetobutylicum. A certain organism, for example, may be described by one investigator as having properties which would bring it within the present-classification, whereas another investigator may report an organism of the same name to have properties which would' definitely exclude it from this classification. It is to be understood, therefore, that my invention includes within its scope the use of any of the the newly devised fermentation conditions to be hereinafter specified, and not to the use of these organisms generally, under any conditions,

The bacteria designated herein as Clostridz'um saccharo-acetobutylicum are described at length 5 in copending application Serial No. 675,459 by J. C. Woodrufi et al., filed June 12, 1933. For the. purposes of the present invention, these bacteria may be briefly defined by the following characteristics: 10

I. Morphological A. Rod-shaped B. Sporesterminal C. Practically indistinguishable from members of the Clostrzdium butyricum group.

II. Biochemical A. Carbohydrate fermentation 1. Inability to produce yields of butyl al-' cohol and acetone consistently greater than 10%, on the weight of the starch, from mashes consisting solely of cereal meal and water 2. Ability to produce yields of butyl alcohol and acetone consistently above-25%, on the weight of the starch, from cereal mashes containing ammonia nitrogen and a suitable neutralizing agent B. Nitrogen metabolism 1. Ability to produce satisfactory yields of butyl alcohol and acetone in starch mashes containing ammonia as the principal source of nitrogen 2. Inability to produce satisfactory yields of butyl alcohol and acetone in starch mashes containing undegraded protein as the sole source of nitrogen 3. Inability to liquefy gelatin or to produce more than slight proteolysis of milk 0. Oxygen requirements 1. Anaerobic-but .will ferment satisfactorily under aerobic conditions due to.

' anaerobic conditions existing within the main body of the fermenting mash D. Temperature range for solvent production 1. From 24 C. to 40 C., preferably 29 C.

to 30 C.

E Hydrogen ion concentration for solvent production in starch mashes 1 Final pH of 4.5-8.5, preferably 5.0-6.2.

The characteristics outlined above may be determined by the usual tests known to those skilled in the art. Suitable methods for this purpose are outlined in copending application Serial No.

675,459, referred to above. With the exceptions noted in the outline, the characteristics were de termined in mashes of commercial concentrations and under optimum fermentation conditions. It will, of course, be recognized by those skilled in the art that the characteristics of this outline refer to normal consistent results and not to abnormally high or low results which may sometimes be obtained with any culture.

Although the present invention includeswithin its scope all bacteria which have in common the characteristics outlined above, irrespective of any differences in minor characteristics not included in this outline, it is preferred to use a particular member of this group of organisms which has been designated Clostridium saccharo-acetobutylicum-alpha. This organism is described below according to the Descriptive Chart of the Society of AmericanBacteriologists.

Name of organism: Clostridium saccharoacetobutylz'cum-alpha Source: Soil I. Morphology 1. Vegetative cells Form: Short and long rods Arrangement: Single and chains Limits of length: 2.5-7.0 microns; of

diameter 1.0-1.3 microns Size of majority: 4.0 x 1.0 microns Ends: Rounded 2. Sporangia: Present Form: Spindled, clavate Limits of length: 2.5-l1.0 microns; of

diameter 1.6-2.3 microns Size of majority: 6.0 x 2.2 microns 3. Endospores: Present Medium used: 20% potato mash; 14

days at 30 C. Stain used: Nigrosin Location of endospores: Central to terminal Form: Ellipsoidal to cylindrical Limits of length: 3.0-5.0 microns; of di'- ameter 1.8-2.2 microns Size of majority: 4.0 x 2.0 microns 4. Motility g In broth: V On' agar: 5. Flagella: Present.

Attachment: Peritrichiate Strain used: Cesares Gil 6. Irregular forms: Many 7. Staining reactions Gram stain: 1 day: Posim e 2 days: Positive, some negative 3 days: Negative, some positive 4 days: Negative 8. Iodine stain: Granulose shown in Clostridia at 48 hours II. Cultural characteristics .1. Agar stroke Medium used: 2% glucose agar containing 0.1% (N'H4)zSO4 Incubation temperature: 30 C. Age: 3 days Growth: Moderate (incubated anaerobically) 7 Form: Beaded Elevation: Raised to convex Luster: Glistening Surface: Smooth to contoured Optical character: Opaque II. Cultural characteristics-Continued III.

Agar stroke-Continued Chromogenesis: None to light cream or light yellow Odor: Butyrous or butylic Consistency: Viscid. Medium: Unchanged Nutrient broth 1% glucose Incubation temperature: 30 C. Age: 2 days Surface growth: None 'Clouding: Yes Odor: Slightly butyrous Sediment: Very slight in 2 days, decided in 7 days Gelatin stab Medium used: 2% glucose gelatin Incubation temperature: 22 C. Age: 30 days Growth: Slight, better at bottom Line of puncture: Beaded Liquefaction: None Degree of liquefaction in 30 days: None Medium: Unchanged Agar colonies Medium used: 2% glucose agar containing 0.1% (NH4)2SO4 Incubation temperature: 30 C. Age: 3 days Growth: Slow Form: Circular Surface: Smooth Elevation: Raised to convex Edge: Entire Internal structure: Finely-granular Physiology Temperature relations Fermentation temperature range: 24-

. Relation to reaction of medium Fermentation pH range: 4.0-7.0

. Chromogenesis Nutrient gelatin: None to slight cream Nutrient agar: None to slight cream Potato: Cream Production of indole Medium: Broth in 1% glucose Test used: Paradimethylaminobenzaldehyde Presence: Absent Production of hydrogen sulfide Medium: Glucose lead acetate agar Presence: Absent Relation to oxygen Litmus milk Reaction: Acid in 1-2 days Acid curd: Slowly formed, 7-14 days Peptonization: Slight in 30 days Reduction of litmus:

Beginning: 1-2 days End: Uncertain due to oxidation of litmus by atmosphere above medium Nitrate reduction Medium: Nitrate-peptone broth III. Physiology-Continued Nitrite: Alpha-naphthylamine-sulphanilic acid 1 Gas: Absent 9. Carbohydrate fermentation Medium used: 1.0% carbohydrate 0.5% peptone I 0.05% KH2PO4 0.05% KzHPO4 0.3% (NH4)2SO4 0.02% MgSO4 .001% NaCl .001% MnSO4 .001% FeSO4 pH adjustedto 6.3 Incubation temperature: 30 C. Time: 72 hours Carbohydrate Acid Gas n sali bl s tsirch Dextrin a Rafilnose Sucrose- Lactosc Mimosa.-- Glucose Levulose. y em-fl 1+ Mannitol Glycerol Dnlcitol n'egative; +very slight; ++moderate; decided; abundant It is known to those skilled in the art that many of the characteristics included in the above descriptive chart are variable and that different results may be obtained by only slight changes in the media, age of culture, or fermentation conditions.- That chart, therefore, is includedherein as an aid in identification of the bacteria and not as an absolute limitation. It is believed that with the aid of the general outline previously ven and the above descriptive chart, one skilled in the art can readily identify Clostridium saccharo-acetobutylicum-alpha with certainty in spite of slight variations in the minor characteristics of the chart. In any event, a check can be obtained by testing the culture at different intervals and under slightly different fermentation conditions noting the characteristics which appear most consistently.

The organisms of this group are widely distributed in nature and may be isolated from such various sources as soil, rotted wood, grain, corn stalks, river mud, and the like. In view of the characteristics listed above, one skilled in the art may readily isolate these organisms from such Sources by known methods of isolation. of course, as is apparent to one skilled in the art, these organisms cannot be isolated from every sample of material tested. However, if a ninnber of different materials are tried, a good culture will nearly always be secured. A suitable method of isolation, for the purposes of the present invention, comprises enrichment in cereal mashes containing ammonia nitrogen and an insoluble neutralizing agent, followed by anaerobic plating if further purification is desired. The procedure outlined in the copending application, herein-- before referred to, will also be found to be satis-.

In general, however, I prefer to utilize corn mash. The concentration of the mash will vary, depending upon the particular source of carbohydrate employed, but, in general, should range between 3 and 6% by weight, calculated on the starch content of the carbohydrate. For example, a mash containing 7% corn (dry weight) will be found to be quite satisfactory.

The nitrogen source constitutes an essential element of my invention and is primarily responsible for the improved'yields secured. The principal source of nitrogen should comprise ammonia, preferably in the form of a salt such as ammonium sulphate'or ammonium phosphate. In addition to the ammonia, it is desirable to include other forms of nitrogen, preferably of the degraded types such as yeast water, steep water, distillery slops, and the like. A certain amount of whole protein may also be found to be desirable, but this is usually present in commercial sources of starchy carbohydrate such as cereal meal or the like. The concentration of ammonia nitrogen'in the mash will depend somewhat upon the amount of degraded nitrogenous material present. In general, however, a concentration of 0.15% to 0.35%, of an ammonium salt will be found to be satisfactory. The following experiment will illustratethe improved results secured by utilizing ammonia as the principal source of nitrogen in this fermentation. In this experiment a mash was prepared containing 7.8% of corn (dry Weight), 0.4% of calcium carbonate, and 0.2% of nitrogenous nutrient. The mash was sterilized in the usual manner, inoculated with an active culture of Clostrz'dium .saccharoacetobutylicum-alpha and incubated at 30. C. for '72 hours. The yields secured for these fermentations, with and without ammonia or corn germ meal, are reported in the table below:

Corn germ Ammonium Ferrnle ntatlon meal Sulphate Yiez ldtwt.h

by wt. by wt. S are It is also essential for obtaining optimum a small amount of a nontoxic insoluble basic neutralizing agent in the mash. Such materials as calcium carbonate, barium carbonate, iron carbonate and the like are suitable for this purpose. Calcium carbonate is to be preferred from anecomonic standpoint and a concentration from 0.2% to 0.6%, on the weight of theltotal mash in excess of that required to neutralize initial acidity, will generally be found to be quite satisfactory. Other methods of securing the hydrogen ion control, such as titrationwith soluble basic materials, may also be employed, but such methods are more delicate and require great skill or expensive apparatus for their satisfactory use; Continuous or semi-continuous electrometric titration is probably the most suitable of these procedures. In any such case, the control should be effected in a manner simulating the action of Solvent ratio 0 S are Butyl Ethyl alcohol alcohol Example II A corn mash containing 7.0% of corn (dry weight), 0.3% ammonium sulphate, and 0.4% of calcium carbonate was prepared and sterilized in the usual manner. The mash was inoculated with 4% by volume of an active culture of the type Clostrzdium saccharo-acetobutylicum which had been isolated from soil. The yield and solvent ratio obtained after incubation at- 30 C. for 72 hours were found to be as follows:

Solvent ratio igiclrl 71;; o stare Butyl Ethyl alcohol Acetone alcohol It is to be understood, of course, that my invention is not to be limited to the specific examples or the specific materials or methods given above. Starchy carbohydrates other than those specifically mentioned may be employed in my process and one skilled in the art can readily make such adjustments in the composition of the media as are necessary to approximate the examples given. Likewise, other sources of nitrogen may be employed, such as amino acids, urea and the like, and as the amount of such lower forms of nitrogen is increased, the amount of ammonia may be correspondingly decreased. One skilled in the art may readily determine, by preliminary fermentations, the optimum concentrations of the various nitrogenous materials employed. In general it may be said that equivalents and modifications of the procedure which would naturally occur to one skilled in the art may be employed without departing 'from the scope of my invention.

My invention now having been described, what I claim is:

1. In a process for the productionof normal butyl alcohol, acetone, and ethyl alcohol by the fermentation of an essentially starchy mash by means of bacteria of the group Clostridium saccharo-acetobutylicum while maintaining the acidity of the mash within the optimum range for fermentation, the improvement which comprises eii'ecting the fermentation in the presence of ammonia nitrogen as the principal source of nitrogen for the bacteria.

2. In a process for the production of normal butyl alcohol, acetone, and ethyl alcohol by the fermentation of an essentially starchy mash by means of bacteria of the group Clostridium saccharo-acetolmtylicum while maintaining the acidity of the mash within the optimum range for fermentation, the improvement which comprises effecting the fermentation in the presence of from 0.15% to-0.35% of an ammonium salt, calculated on the total weight of the mash.

3. In a, process for the production of normal butyl alcohol, acetone, and ethyl alcohol by the fermentation of an essentially starchy carbohydrate mash by means of bacteria of the group Clostridium saccharo-acetobutylicum, the improvement which comprises effecting the fermentation in the presence of from 0.15% to 0.35% of an ammonium salt, calculated on the total weight .of the mash, and from 0.2% to 0.6% of a nontoxic insoluble basic neutralizing agent in excess of that required to neutralize any initial acidity of the mash.

4. In a process for the production of normal butyl alcohol, acetone, and ethyl alcohol bythe fermentation of corn mash by means of bacteria of the group Clostridium saccharo-acetobutylz'cum, the improvement which comprises effecting the fermentation in the presence of from 0.15% to 0.35% of ammonium sulphate, calculated on the total weight of the mash, and from 0.2% to 0.6% of calcium carbonate in excess of that required to neutralize any initial acidity of the mash.

5. A process for the production of normal butyl alcohol, acetone, and. ethyl alcohol which comprises subjecting a mash containing starchy carbohydrate as the principal fermentable carbohydrate and ammonia nitrogen as the principal source of nitrogen to the action of bacteria of the group Clostrz'dium saccharo-acetobutylicum, at temperatures from 24 C. to 40 C., while controlling the acidity of the mash during the fermentation whereby the final hydrogen ion concentration secured by the action of the bacteria falls within the range pH 5.0 to pH 6.5.

6. A process for the production of normal butyl alcohol, acetone, and ethyl alcohol which comprises subjecting a mash containing starchy carbohydrate as the principal fermentable carbohydrate and ammonia nitrogen as the principal source of nitrogen to the action of bacteria of. the group Clostridium saccharo-acetobutylcium, at temperatures from 24 C. to 40 C., while controlling the acidity of the mash during the fermenta ion by means of a nontoxic insoluble ,basic neutralizing agent whereby the final hydrogen ion concentration secured by the action of the bacteria falls within the range pH 5.0 to pH 6.5.

7. A process for the production of normal butyl alcohol, acetone, and ethyl alcohol which comprises subjecting the mash containing starchy carbohydrate as the principal fermentable carbohydrate and an ammonium salt as the principal source of nitrogen to the action of bacteria of the group Clostridium saccharo acetobutylicum, at temperatures from 24 C. to 40 C., while controlling the acidity of the mash during the fermentation by means of calcium carbonate whereby the final hydrogen ion concentration secured by'the action of the bacteria falls within the range pH 5.0 to pH 6.5.

8. A process for the production of normal butyl alcohol, acetone, and ethyl alcohol wh ch comprises subjecting corn mash containing from 0.15% to 0.35% of ammonium sulphate to the action of bacteria of the group Clostrz'dium saccharo-acetobutylicum, at temperatures from 24 C. to 40 C., while controlling the acidity of the mash during the fermentation by means of calbutyl alcohol, acetone, and ethyl alcohol which cium carbonate whereby the final hydrogen ion concentration secured by the' action of the bacteria falls within the range pH 5.0 to pH 6.5.

9. In a process for the production of normal butyl alcohoL-acetone, and ethyl alcohol by the fermentation of. an essentially'starchy carbohydrate mash by means of Clostridium saccharoacetobutylicuml-alpha, the improvement which comprises eifecting 'thei'erm'entation in the presence offrom-0.15%.to 0.35%. of an ammonium salt, calculated on the total" weight of the mash,

andfrom 0.2% to 0.6% of a nontoxic insoluble basic neutralizingagent in excess of that required to neutralize any initial acidity: of the mash.

-10. In a process for the production of normal butyl alcohol, acetone, and ethyl alcohol by the fermentation of corn mash by means of Clostridium saccharo-acetobutylicum-alpha, the improvement which comprises effecting the fermentation in the presence of from 0.15% to 0.35% of ammonium sulphate, calculated on the total weight of the mash, and from 0.2% to 0.6% of calcium carbonate in excess of that required to neutralize any initial acidity of the mash.

11. A process for the production of normal butyl alcohol, acetone, and ethyl alcohol which comprises subjectinga mash containing starchy carbohydrate as the principal termentable carbohydrate and ammonia nitrogen as the principal source ofnitrogen to the action of Clostridium saccharo-acetobutylicum-alpha, .at temperatures from 24 C. to C.,- while controlling the acidity of the mash during the fermentation whereby the final hydrogen ion concentration secured by the action of the bacteria falls within the range pH 5.0 to pH 6.5.

12. A process for the production of normal comprises subjecting a mash containing starchy carbohydrate as the principal fermentable carbohydrate and ammonia nitrogen as the principal source of nitrogen to the action of Clostridz'um saccharo-acetobutylicum-alpl'a, at temperatures from 24 C. to 40 C., while controlling the acidity of the mash during the fermentation by means of a nontoxic insoluble basic neutralizingagent whereby the final hydrogen ion concentration secured by the action of the bacteria falls within the range pH 5.0 to pH 6.5.

13. A process for the production of normal butyl alcohol, acetone, and ethyl alcohol which comprises subjecting the mash containing starchy carbohydrate as the principal fermentable carbo hydrate and an ammonium salt as the principal source of nitrogen to the action of. Clostridium saccharo-acetobutylicum-alpha, at temperatures irom 24 C. to 40 C., while controlling the'acidity of the mash during the fermentation by means of calcium carbonate whereby the final hydrogen ion concentration secured by the-action of the bacteria'falls. within the range pH 5.0 to pH 6.5.

1-4. A process for the production of normal butyl alcohol, acetone, and ethyl alcohol which comprises subjecting corn mash containing from- 

